Immune response, oxidative stress, and histological changes of Wistar rats after being administered with Parascaris equorum antigen

Worldwide, particularly in developing nations, helminth infections are the leading causes of livestock illness and mortality. Parascaris (P.) equorum, a parasitic worm from the Ascarididae family, significantly impacts the production, health, and working performance of equines. This study aimed to investigate the impact of intraperitoneal sensitization of P. equorum on the immune system, oxidative stress, and histology in Wistar rats. After acclimatization for 7 days, we divided the rats into five groups, each consisting of six rats. Group I, serving as the control, was administered distilled water, followed by groups II (day 7), III (day 14), IV (day 21), and V (day 33). The rats were euthanized every day mentioned (Days 7–33). On day 0, a dosage of 1ml/100 gm rat (containing 500 μg/ml protein content) emulsified crude antigen extract with an incomplete Freund’s adjuvant (1:1 volume), followed by a second dose of the same antigen concentration on day 7. To assess the allergenicity of this nematode, we measured a whole blood profile, serum levels of IFN-γ, IL-5, IL-10, IL-13, and IL-33, total immunoglobulins IgE and IgG, and oxidative stress markers. Also, we examined histological changes in the liver, kidney, and spleen. The results showed that values of total leukocyte count, granulocytes, monocytes, and lymphocytes were significantly (P < 0.05) increased on day 14 post-infection relative to other days of investigation. It was found that the levels of total immunoglobulins (IgE and IgG) and cytokines (INF-γ, IL-5, IL-13, and IL-33) on days 14 and 21 were significantly higher than in the control group. At all periods of the experiment, the injected group exhibited significantly higher concentrations of MDA and NO compared to the control group (P < 0.05). Conversely, GSH and CAT levels (P < 0.05) dropped significantly on days 7, 14, and 21. Different rat tissues showed alterations. Ultimately, this study described the detrimental effects of P. equorum crude antigen administration on the immune system, oxidative states, and histological changes of Wistar rats at various intervals.


Worm collection and parasitological study
The alimentary tracts of domestic horses (Equus ferus caballus Linnaeus 1758; Family Equidae) were necropsied for educational purposes at the Faculty of Veterinary Medicine, Cairo University, Egypt.Each organ of the tract was isolated, placed in separate shallow plastic jars filled with normal saline (0.85%), and then transported to the Parasitology laboratory in the Zoology Department of the Faculty of Science.As described by Boomker et al. 18 , the contents of the intestine and stomach were transferred to distinct plastic containers filled with water.We thoroughly combined the contents and the mixture using a glass pipette.Different portions were placed in petri dishes and observed under a dissecting microscope.The retrieved worms were rinsed in physiological saline and 10% acetic acid multiple times to eliminate residual mucus or host debris.The recovered worms were washed in physiological saline and then in 10% acetic acid to remove any mucus or host debris.Five to ten millimeters of the cephalic and caudal extremities were cut off with a blade and rinsed in a lactophenol solution to prepare individual worms for light microscopy.Photomicrographic images were obtained by a LEICA DM 750 microscope equipped with a LEICA ICC 50 HD camera.Worms were subjected to SEM according to the following procedures: fixation in a 3% glutaraldehyde solution, subsequent washing in a 0.1M sodium cacodylate buffer (pH 7.4), dehydration via a graduated ethanol series (50%, 60%, 70%, 80%, 90%, and 100%), and drying at 30 °C for 30 min utilizing a critical point drier (LEICA, EM CPD300).Using an accelerating voltage of 25 kV, dried specimens were examined with a JEOL JSM-5200 SEM (Tokyo, Japan) after being mounted on stubs and coated with gold.Body dimensions were expressed as means (mm ± S.E).The parasite species were identified according to the keys of Lichtenfels 19 .

Preparation of nematode crude antigen
Nematode worms (3-5) were homogenized in PBS solution by a pestle homogenizer.10-20 µl of a protease inhibitor mixture was added to the worm homogenate.The suspension was centrifuged for 20 min at 10,000 rpm

Assessment of oxidative stress markers
Hepatic rat tissues from all tested groups were isolated and homogenized (10% wt/vol) in an ice-cold 0.1 M Tris HCl buffer (pH 7.4), centrifuged for 10 min at 3000 rpm, and the resulting supernatant was kept at -20°C until used.The lipid peroxidation marker, malondialdehyde (MDA), was assessed as outlined by Buege and Aust 24 , and nitric oxide (NO) was determined according to Montgomery and Dymock 25 .The activity of the antioxidant enzyme catalase (CAT) was determined as described by Aebi 26 , and the concentration of glutathione reduced (GSH) estimated based on the method described by Beutler et al. 27 .Oxidative stress biomarker levels were calculated using reagent kits purchased from Bio-Diagnostic (Egypt).

Histopathological investigation
Different rat tissues (Liver, kidney, and spleen) were isolated, cleaned with physiological saline, and fixed with 10% formalin for 24 h.Following that, as described by Nanji et al. 28 , the samples were dehydrated in an ascending series of alcohol concentrations, treated with xylene for clarity, embedded in paraffin, and then sectioned to a thickness of 5 µm.Hematoxylin and eosin (H&E) were used to stain the sections before histologic examination under a light microscope to detect histological changes such as congestion, hemorrhage, cell necrosis, edema, and hepatocyte vacuolization.

Statistical analysis
Data were expressed as means ± standard error of the mean (SEM) for six animals in each group.One-way analysis of variance (ANOVA) was employed to assess the differences between groups, followed by a Duncan post hoc test using SPSS version 20 (SPSS Inc., Chicago, IL, USA) software.P values less than 0.05 were regarded as statistically significant.

Ethical approval and consent to participate
The current study was conducted in accordance with the relevant guidelines and regulations of ARRIVE guidelines.The Cairo University Institutional Animal Care and Use Committee (CU-IACUC) approved all experimental procedures using animals in this study under the relevant document (No.CU/I/S/44/16).All laboratory animal use procedures in this study were agreed upon according to the Ethics of Research Committee regulations at the Faculty of Science, Cairo University, and received the approval number (No. CU/I/S/44/16).

Morphological description of the nematode worms (based on 5 specimens, Figs. 1 and 2)
At the macroscopic level, the worms retrieved from the gastrointestinal tract of the dissected horse specimen exhibited elongated, white, and unsegmented characteristics.With the naked eye, male and female worms were distinguishable by their wrapped and upright ends.Males measured 15 ± 2 (12-17) cm in length, while females measured 17 ± 2 (14-20) cm.Light and scanning electron micrographs revealed that both sexes have broad anterior ends with three interlocked lips that resemble a shamrock, two sub-ventral and one dorsal, and two or more cephalic papillae surrounding the mouth opening.Deep post-labial constriction separated the labia from the rest of the body, leaving a triangular-shaped inter-labia in between.The cuticle was transversely striated and contained prominent, narrow annules.There were no caudal alae seen.The male tail was long and characterized by a triangular shape and a pointy tip, adorned with many papillae organized in longitudinal rows supplied with two spicules.At the same time, the female had a conical tail that was somewhat attenuated at the distal third.

Hematologic findings
As demonstrated in Table 1, values of total leukocyte count and differential cells, granulocytes, monocytes, and lymphocytes were considerably (P < 0.05) raised on day 14 post-injections relative to other days.Hemoglobin content declined on day 14, and red cell count and hematocrit increased significantly on days 7, 21, and 33.Additionally, MCV revealed a significant increase on day 7.While MCH revealed no significant change throughout the entire day of investigation, in terms of platelet count, it was relatively lower on day 7 compared to the control but significantly higher on days 14, 21, and 33.MPV significantly increased on days 7, 14, and 21 while returning

Immunological profile
The current study used the ELISA technique to identify the humoral immune responses against the experimental infection of Wistar rats with P. equorum crude antigen extract.Table 2 indicated that the levels of IFN-γ, IL-5, and IL-13 were significantly higher (P < 0.05) in the injected groups on days 14 and 21 compared to the control rats.However, these levels returned to normal on day 33 following the injection.IL-33 was significantly (P < 0.05) elevated on days 7, 14, and 21.Meanwhile, IL-10 showed no significant change among all the examined groups.The concentrations of total IgE were significantly higher (P < 0.05) on days 14, 21, and 33 compared to the control group.Similarly, the levels of total IgG considerably increased (P < 0.05) on days 14 and 21 and recovered to average values on day 33 post-injection.

Oxidative stress biomarkers
Figure 3 revealed that the intraperitoneal sensitization of Wistar rats by P. equorum crude antigen led to oxidative stress in the hepatic tissue, as evidenced by a notable increase in MDA and NO levels in the injected group compared to the control group (P < 0.05) through all different time intervals post-infection.Also, a substantial (P < 0.05) decrease in GSH and CAT concentration was detected on days 7, 14, and 21 post-injections, returning to a normal state at day 33, like the control.

Histopathological findings
Microscopic examination of the liver of control rats appeared normal, with orderly arranged hepatocytes in normal lobular architecture (Fig. 4a).In contrast, on day 7, liver sections of injected rats showed a multifocal area of coagulative necrosis of the hepatic parenchyma associated with the inflammatory cell aggregations.(Fig. 4b).On day 14, the liver tissue showed marked portal fibroplasia associated with bile ductular epithelial hyperplasia (Fig. 4c).The multifocal areas of mononuclear inflammatory cell aggregations in the hepatic parenchyma and hemorrhages were frequently detected on day 21 (Fig. 4d).Finally, on day 33 post-injection, the microscopic examination of the liver sections also showed multifocal hepatitis areas characterized by the accumulation of mononuclear inflammatory cells in the hepatic lobules and the portal areas, accompanied by severe hemorrhagic areas with excessive erythrocyte exudation replacing the hepatic parenchyma (Fig. 4e).
Compared to the kidney tissue of control rats, which displayed a normal structure of both the renal cortex and medulla (Fig. 5a), kidney sections on day 7 following the injection showed fewer necrobiotic changes in the epithelial lining cortical renal tubules, with numerous casts in the lumen of several renal tubules (Fig. 5b).On day 14, it was observed that the renal tubules suffered from coagulative necrosis (Fig. 5c).Necrobiotic changes www.nature.com/scientificreports/were still observed in the renal tubules with congestion on day 21 (Fig. 5d).On day 33, necrosis of renal tubules, congestion, and renal casts still observed (Fig. 5e).
The splenic tissue of control rats exhibited a normal histology of splenic parenchyma formed of white and red pulps; splenic cords and sinusoids represented the red pulp, while the white pulp is composed of three compartments: the periarterial lymphatic sheath (PALS), lymphatic follicles, and the border zone (Fig. 6a).On the other hand, at day 7, splenic tissues showed some histopathological alterations, including the expansion of red pulps accompanied by mild atrophy of splenic follicles and existence of a variable number of megakaryocytes in some sections (Fig. 6b).Also, on day 14, perivascular edema was frequently detected in splenic tissue, accompanied by an increased number of inflammatory cell infiltration (Fig. 6c).On day 21, in addition to the expanded red pulps affected splenic white pulps, numerous congested blood vessels were noticed (Fig. 6d).On day 33, the splenic capsule showed severe thickening with abundant fibroplasia and intense mononuclear inflammatory cell infiltration (Fig. 6e).

Discussion
Gastrointestinal parasitism has been identified as one of the most significant threats to equids in developing countries 29 .Parascaris equorum is a common ascarid nematode that inhabits the small intestines of equines, mainly young animals of horses and donkeys, which are highly susceptible for infection 30 .Using light and scanning electron microscopy, the morphological identification of the worm recovered in the current work indicated that it belongs to P. equorum by having a distinctive head region characterized by a shamrock-like three interlocked lips.This is consistent with the findings of studies published by Lichtenfels 19 .Additionally, Pilitt et al. 31 noted that the labial and cuticular morphologies, the size and shape of the lip, and the spacing of the denticles surrounding the lip margins are the most valuable characteristics for differentiating morphologically similar developmental stages.Similar to the descriptions given by Morsy et al. 32 , it was observed that the cuticle of the current P. equorum is finely striated, containing a narrow annulus devoid of markings.Furthermore, the male possesses a shorter tail than the female, distinguished by two spicules of equivalent length.Phasmids were observed at the caudal end of females.Several studies have documented this severe parasite's morphological and morphometric description affecting foals and weanlings worldwide 7,33,34 .
Immunity during gastrointestinal parasite infections involves both humoral and cellular responses 35 , which are known to be closely dependent 36 .Although gastrointestinal nematode infections are globally prevalent, there is limited understanding of the specific mechanisms that generate protection against them 37 .The mechanisms underlying resistance to infection vary significantly between hosts and parasites 38 .However, the polarized type 2 response (IL-4, IL-5, IL-9, and IL-13) might reduce the severity of infection and contribute to the nematode parasite's eradication 10 .IFN-γ is mainly secreted by activated T cells and natural killer (NK) cells and is considered  the chief modulator of innate and adaptive immunity 39 .The current work demonstrated that crude antigen P. equorum promoted a significant rise (P < 0.05) of IFN-γ levels on days 14 and 21 post-injection while returning to normal values on day 33 compared to control rats.This finding is corroborated by Shlash et al. 40 , which reported higher levels of IFN-γ in A. duodenale infected patients.IL-5 is produced by Th2 cells to stimulate antibody production from activated B cells after stimulation with allergens 41 .On days 14 and 21 following injection, it was observed that IL-5 significantly (P < 0.05) increased.In contrast, Tran et al. 42 reported that Nippostrongylus brasiliensis infection induces eosinophilia and IL-5 production after 7 days, and parasite expulsion occurs after a rapid decline of IL-5 after 14 days.IL-13 possesses significant anti-inflammatory characteristics and is regarded as a pivotal cytokine in regulating pathogens, including helminthic parasites and allergic and inflammatory disorders 43 .Our data revealed that P. equorum crude antigen caused a significant increase in IL-13 levels on days 14 and 21 post-injection relative to other groups.IL-33 is an anti-inflammatory cytokine and a potent stimulant of the innate immune system and plays a role in various diseases 44,45 .It is released when intestinal nematode larvae infect epithelium and cause damage as they move through the lungs and into the airways 46 .The level of IL-33 was linked to the number of worms 47 ; lower levels of IL-33 in immunized mice were related to fewer lung larvae, less inflammation, and better function.Also, Humphreys et al. 48reported that IL-33 significantly contributes to eliminating bacterial and parasitic www.nature.com/scientificreports/diseases.In addition, Pastorelli et al. 49 reported that animal models of ulcerative colitis show elevated levels of IL-33.Thus, the current IL-33 high level of the injected groups on days 7, 14, and 21 suggested that it is a sign of protection against P. equorum crude antigen infection.In a similar vein, Neill et al. 50reported that nematode infection increased IL-33 levels.The cytokine IL-10 has crucial roles in modulating immune and inflammatory processes, which regulate inflammation by preventing the production of monocytes, macrophages, and numerous other proinflammatory cytokines and chemokines that stop inflammation-related tissue damage 51 .In the present study, IL-10 did not exhibit a significant difference among the groups, which explains the prominently observed inflammation in all different tissues.The utilization of IgE and IgG immunoglobulins is critical in diagnosing various pathological states linked to bacterial, viral, and parasitic infections, allowing for the implementation of the correct treatment strategies 52 .There was a notable increase (P < 0.05) in the overall IgE and IgG concentrations of infected rats on days 14 and 21 compared to the control group.These results are consistent with Wright and Bickle 53 , who reported a rise in hookworm-specific IgG and IgE levels in humans following infection.The production of IgE is predominantly mediated by interleukins IL-4, IL-10, and IL-13 via the induction of B-cell switching 54 .Hence, the substantial increase in IgE levels in infected rats may be linked to elevated IL-13 levels rather than the examined IL-10, which did not exhibit any significant variation among the groups.As suggested by Cetre et al. 55 and Mostafa et al. 56 , the elevated concentration of total IgG observed might be linked to the generation of INF-γ.
Parasitic infection can trigger oxidative stress, resulting in elevated ROS generation and reduced antioxidant levels 57 , which can damage membranes, DNA, and protein structures 58 .Overproduction of ROS leads to elevated levels of malondialdehyde (MDA), the primary product of lipid peroxidation 59 .It is considered the primary process by which ROS causes tissue damage and serves as a biomarker for oxidative stress 60 .Furthermore, inflammatory responses are accompanied by ROS production, which can impair vital functions and cause cellular death 61 .Compared to the control group, the current study revealed a substantial increase in MDA and NO concentrations on days 14, 21, and 33 following P. equorum crude antigen injection in rats, confirming that these nematode species induce oxidative stress and inflammation in accordance with a study conducted by Attia et al. 29 .CAT is the essential enzyme in reducing oxidative damage caused by free radicals; it contributes to the reduction of hydrogen peroxide to oxygen and water 62,63 .Intestinal parasitic infection inhibits the antioxidant system by lowering GSH levels.This drop was linked to the higher cytotoxicity of the generated H 2 O 2 caused by the inhibition of glutathione reductase, which keeps glutathione in a reduced state 64 .Additionally, ROS generated by the parasites depletes the host's catalase (CAT) and glutathione (GSH) 65 .In this study, P. equorum antigen extract exerted a substantial (P < 0.05) suppressive impact on the level of CAT and GSH on days 7, 14, and 21 post-injection.These findings agreed with Derda et al. 66 , who noted that T. spiralis resulted in the breakdown of antioxidant enzyme systems in infected mice, disrupting metabolite balance through increased lipid peroxidation.Da Silva et al. 67 also reported that T. evansi infection in camels reduced GSH content.Thus, it seems that the effect of intraperitoneal administration of this nematode antigen reflects an increase in oxidative stress related to intestinal nematode infection 68 .
Histopathological investigations displayed that P. equorum crude antigen extract caused severe changes in the infected rat tissues, including the liver, kidneys, and spleen, demonstrating a similar pattern of changes with time over days of infection.Liver sections showed inflammatory cell aggregations and portal fibroplasia associated with bile ductular epithelial hyperplasia.Moreover, hemorrhages were frequently detected replacing the hepatic lobules, and mononuclear inflammatory cell aggregation in the hepatic lobules and the portal areas, accompanied by severe hemorrhagic areas, was observed.These findings are consistent with Minemura et al. 69 who report that the liver, when subjected to various infections caused by hepatotropic and non-hepatotropic pathogens, can enter the liver through the systemic and portal circulation.These pathogens can initiate liver damage, leading to diverse manifestations.Sections of the kidney showed less necrotic changes in the epithelial lining of the cortical renal tubules and more casts in the lumen of many of the renal tubules.It was observed that renal tubules suffered from coagulative necrosis.Necrobiotic changes were also observed in the affected renal tubules with congestion.Barsoum 70 demonstrated that parasitic infections can cause kidney impairment.
The infected rats' splenic tissue showed some histopathological alterations whereas the red pulps became more prominent, the splenic follicles became smaller, and there were different numbers of megakaryocytes.Perivascular edema was frequently detected, accompanied by an increased number of inflammatory cell infiltrations.Congested blood vessels were noticed, along with mononuclear inflammatory cell infiltration.The splenic capsule showed severe thickening, abundant fibroplasia and intense mononuclear inflammatory cell infiltration.Adhesion between the splenic parenchyma and adjacent organs was detected in several cases.These findings align with those of Fahmy and Diab 71 who observed abnormalities in splenic lymphoid tissue associated with nematode infestation.Moreover, Fahmy and Diab 71 and Ahmed et al. 72 documented that tissue damage and inflammation were caused by elevated levels of reactive oxygen species (ROS) in infected rats.All the above-mentioned pathological alterations in rat tissues may be related to oxidative stress enhancement in histopathological sections.

Conclusions
From the current study, it was concluded that sensitizing Wistar rats with P. equorum crude antigen extract led to inflammatory immune responses via increasing total leukocyte count on day 14 post-infection compared to other days of investigation and stimulating the production of antibodies, IgE and IgG, Th1-related cytokine (IFN-γ), and Th2 cytokines (IL-5, and IL-13), and innate immune responses (IL-33) particularly at days 7 and 14 post-injection.Furthermore, antioxidant markers, CAT, and GSH were downregulated, and elevated MDA and NO levels were observed on days 7, 14, and 21 following injection, indicating an oxidative stress state.Consequently, these conditions resulted in severe alterations in various histological sections, including the liver, kidney, and spleen, during the time period of the study.These findings provide a valuable screening perspective on the

Figure 1 .
Figure 1.Photomicrograph of adult nematode, P. equorum cleared with lactophenol.(a, b) Anterior extremity of worm showing 3 lips (L) one dorsal, and two subventrals forming interlabia (IL) in between and separated from the body by a deep post-labial constriction (PC), mouth opening (MO), and muscular esophagus (OE).(c, d) Transverse striations (TS) of the body cuticle sharply defined.(e) The female has a conical tip.(f) Male posterior ends with two short spicules (SP).

Figure 2 .
Figure 2. Scanning electron micrographs of P. equorum.(a) The adult worm's anterior end displayed three interlocked lips (L), which were organized in the form of one dorsal and two sub-ventrals around the mouth opening (MO) and creating an inter-labium (IL) in between.(b) Transverse striations of the cuticle (TS).(c) Button-like phasmid (PH).(d) Conical end of a female with a prominent anus (A).

Figure 3 .
Figure 3. Hepatic levels of CAT, GSH, MDA, and NO in rat groups injected with P. equorum crude antigen on days 7, 14, 21 and 33 post-injections compared to control group.Data is presented as mean ± SEM (n = 6), where values with different superscript letters are regarded as statistically significant (P < 0.05).

Figure 4 .
Figure 4. Photomicrographs of liver sections of Wistar rats.(a) The control group showed normal healthy polygonal hepatocytes (H) with rounded vesicular nuclei and acidophilic cytoplasm.(b-e) Intraperitoneal injected rats investigated at different time points, (b) Group II (day 7) showing vacuolated hepatocytes with aggregation of inflammatory cells replaced necrotic hepatocytes (arrow).(c) Group III (day 14) showing portal fibrosis with ductular epithelial hyperplasia.(d) Group IV (day 21) showing severe hepatic hemorrhage.(e) Group V (day 33), focal portal hepatitis was detected.All sections were stained with H&E, magnification 400x, and a scale bar of 25µm.

Figure 5 .
Figure 5. Photomicrograph of kidney sections of Wistar rats.(a) The control group showed normal renal tubules in the cortex.(b) On day 7, injected rats showed casts in the renal tubules (arrows).(c) On day 14, injected rats showed severe necrosis of renal tubular epithelium (head arrows).(d) On day 21, injected rats showed congestion of the renal cortex.(e) On day 33, injected rats showed renal casts (arrows).All sections were stained with H&E, magnification 400x, and a scale bar of 25 µm.

Figure 6 .
Figure 6.Photomicrographs of the splenic sections of Wistar rats.(a) Control group showing normal lymphoid tissue (white pulp) with germinal center (arrow).(b) On day 7, the injected group showed a variable number of megakaryocytes (arrows).(c) On day 14, injected rats showed inflammatory cell infiltration (arrow).(d) On day 21, injected rats showed congested blood vessels.(e) On day 33, injected rats showed severe thickening of the splenic capsules (asterisks) with excessive fibrosis and inflammatory cell infiltration.All sections stained with H&E, magnification 400x, and scale bar: 25 µm.

Table 1 .
Complete blood analysis of rats intraperitoneally injected with crude antigen extract of P. equorum at different time points compared to control.Data is presented as mean ± SEM (n = 6), where values with different superscript letters are regarded as statistically significant (P < 0.05).WBCs, white blood cells; Gran, Granulocytes; Mon, Monocytes; Lym, Lymphocytes; Hb, hemoglobin; RBCs, Red blood cells; PCV, packed cell volume; MCV, mean cell volume; MCH, mean cell hemoglobin; MPV, mean platelet volume; PDW, red cell distribution width; PLCC, Platelet-large cell count.